Plasma autoantibody biomarkers for basal like breast cancer

ABSTRACT

Cancer patients make antibodies to tumor-derived proteins that are potential biomarkers for early detection. Twenty-eight antigens have been identified as potential biomarkers for the early detection of basal-like breast cancer (Tables 1, 2). Also, a 13-AAb classifier has been developed that differentiate patients with BLBC from healthy controls with 33% sensitivity at 98% specificity (Table 3).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/743,906, filed Jan. 15, 2020, which claims priority to InternationalPatent Application No. PCT/US2015/064792, filed Dec. 9, 2015, whichclaims priority to U.S. Provisional Patent Application No. 62/089,631filed on Dec. 9, 2014, the disclosures of which are incorporated byreference herein in their entireties.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under U01 CA117374awarded by the National Institutes of Health. The government has certainrights in the invention.

TECHNICAL FIELD

The disclosure herein related to biomarkers for cancer and moreparticularly to autoantibody biomarkers for breast cancer.

BACKGROUND

Despite significant improvement in early detection by routinemammography, breast cancer remains a global challenge. Current screeningmammography only detects 70% of breast cancers. Breast tumors associatedwith high breast density and highly proliferative cancers are frequentlynot detected by screening. Therefore, there is an urgent need forbiomarkers that can detect potentially invasive breast cancer in theirearly stages.

Like other cancers, breast cancer is heterogeneous, comprising multiplemolecular subtypes with unique characteristics of prognosis, response totreatment and risk of recurrence. This heterogeneity affects thebiomarker discovery, requiring both larger sample sizes and differentstatistical approaches from traditional methods of evaluation. Abiomarker (or panel) may perform well for one molecular subtype but notanother.

Current screening mammography also has limitations in detecting thebasal-like subtype cancers. The fraction of BLBC within all breastcancer cases detected by mammography is less than its natural frequencyin the general population, indicating that BLBC was under-diagnosed byroutine mammography and more frequently detected as interval breastcancer. Early detection of BLBC by screening mammography is compromisedpartly by its high proliferation rate and lack of suspicious features ofregular breast malignancy, such as microcalcifications, irregularspiculated masses or pleomorphic microcalcifications. Moreover, BLBC isoften present in women less than 50 years old, who are not recommendedfor routine mammogram by US Preventative Service Task Force mammogramguidelines.

Considering the high frequency of disease in women younger than 50 yearsold, a potentially large population with BLBC does not benefit frompresent breast cancer screening and would benefit from a molecular testfor the disease. Therefore, there is an urgent need for biomarkers thatcan detect potentially invasive basal like breast cancer in their earlystages.

SUMMARY

We have identified 28 antigens as potential biomarkers for the earlydetection of basal like breast cancer. These biomarkers were selectedout of 10,000 tumor antigens in a sequential screening study and yieldedsupporting evidence in a blinded validation study. These biomarkersshould be useful components of diagnostic tests and personalizedtherapeutics for breast cancer.

All references cited throughout are hereby incorporated by referenceherein.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts dotplots of AAb responses in various breast cancersubtypes.

DETAILED DESCRIPTION

Notably, the sensitivity of a subtype-specific biomarker can never behigher than the prevalence of that subtype in the population where it istested. The ability to find biomarkers for cancer detection with highsensitivities has likely been impaired by this effect. This isespecially true for disease subtypes that are less common in the overallpopulation. Basal like breast cancer is a breast cancer subtype withaggressive disease progression and poor prognosis. It overlapssignificantly with triple negative breast cancer (TNBC), a clinicalpathological subtype characterized by negative tissue staining ofestrogen receptor (ER), progesterone receptor (PR), and the absence ofhuman epidermal growth factor receptor 2 (HER2) amplification.

TNBC is associated with African American ethnicity, younger age,advanced stage at diagnosis and poorer outcomes. However, TNBC itselfhas been found to be molecularly heterogeneous in two recent studies.Among patients with TNBC, a core basal subtype with expression ofepidermal growth factor receptor (EGFR) or basal cytokeratin 5/6 (CK5/6)characterized a group of patients with worse prognosis than the rest ofthe patients with TNBC. This five marker subtyping (ER-, PR-, HER2-, andeither EGFR+or CK5/6+) is highly correlated with BLBC defined by geneexpression profiling.

Current screening mammography has limitations in detecting thebasal-like subtype. The fraction of BLBC within all breast cancer casesdetected by mammography is less than its natural frequency in thegeneral population, indicating that BLBC was under-diagnosed by routinemammography and more frequently detected as interval breast cancer.Early detection of BLBC by screening mammography is compromised partlyby its high proliferation rate and lack of suspicious features ofregular breast malignancy, such as microcalcifications, irregularspiculated masses or pleomorphic microcalcifications.

Moreover, BLBC is often present in women less than 50 years old, who arenot recommended for routine mammogram by US Preventative Service TaskForce mammogram guidelines. Considering the high frequency of disease inwomen younger than 50 years old, a potentially large population withBLBC does not benefit from present breast cancer screening and wouldbenefit from a molecular test for the disease.

In practical terms, molecular diagnostic tests for the early detectionof cancer should be performed on readily accessible samples, likeplasma, because they are likely to be performed on many individuals. Theconcentration of many cancer biomarkers in blood tends to be very lowbecause it relies upon secretion by cancer cells which are few in numberin the pre-clinical stage. Moreover, the biomarker gets diluted in alarge volume of plasma volume and only a fraction of the secretedbiomarker gets distributed to the plasma.

An alternative strategy is to exploit the ability of the immune systemto detect the presence of tumor cells through the generation ofautoantibodies. These responses of the adaptive immune system againsttarget tumor antigens amplify the signals from the minute amount oftumor proteins released from cancer tissue.

We have previously conducted an autoantibody biomarker discovery forbreast cancer on our nucleic acid programmable protein array (NAPPA)platform. NAPPA allows us to display thousands of freshly produced fulllength human proteins on glass slides without the need of proteinpurification and has been applied to the study of disease specificantibodies in diseases ranging from infectious to autoimmune to cancers.In that study, we took a three stage study design to identifyautoantibody markers from 4988 human proteins.

The goal for the discovery stage 1 was to eliminate non-reactive anduninformative (i.e. no difference between case and control) antigens andreduce the total number of antigens. The top 761 antigens were selectedbased on differential reactivity between cases and controls. The goalfor the training stage II was to identify candidate autoantibodymarkers. The goal for the blinded validation stage III was to validatepotential biomarkers. This yielded a panel of 28 markers showingsensitivities in the 10-30% range with specificities from 80-100%.

However, the sample cohort used in this study was a mixed population ofpredominantly women with ER+PR+breast cancer. Therefore, the utility ofthese markers in subtypes like BLBC or Her2+ are likely limited,considering their relatively low percentage among breast cancerpatients. Here, we aimed to identify BLBC specific autoantibodies byprofiling humeral immune responses of BLBC patients against 10,000 humanproteins.

One challenge associated with the use of “omics” technology to study ahomogeneous cancer subtype is the requirement of a large number ofsamples to have sufficient power of analysis. To this end, our study wassupported by the Polish Breast Cancer Study, in which over 2386 breastcancer patients were enrolled.

Paraffin embedded tissue samples were collected for immunohistochemical(IHC) analysis and disease classification. We successfully collectedplasma samples from 145 patients classified to be basal-like subtype byeither PAM50 gene signature based on mRNA expression profiling, ortissue IHC staining of ER, PR, HER2, EGFR and CK5/6. In addition, foreach patient, we also collect plasma sample from an age, gender andlocation matched healthy donor. Here, we first profiled sero-reactivityagainst 10,000 human proteins in 45 BLBC patients and 45 matchedcontrols.

Candidate antigens were selected, assayed for their autoantibodies inBLBC using customized NAPPA and enzyme-linked immunosorbant assay(ELISA), and validated using an independent patient cohort in a blindfashion. See, for example, “Tracking humoral responses using selfassembling protein microarrays,” Proteomics Clin Appl. 2008 Oct2(10-11):1518-27. A biomarker signature was also developed todiscriminate basal-like tumors from age and location matched healthyindividuals. We further evaluated the specificity of the panel ofautoantibodies to basal-like tumors using a set of patients with otherbreast cancer subtypes.

The 28 antigens that we have identified as potential biomarkers for theearly detection of basal like breast cancer are (Table 1, Table2): P53(TP53), NY-ESO-1 (CTAG1B), NY-ESO-2 (CTAG2), RNF216, PPHLN1, PIP4K2C,ZBTB16, TAS2R8, WBP2NL, DOK2, PSRC1, MN1, TRIM21, POU4F1, SSMEM1, LMO4,BCL2, KRT8, TSGA13, PVRL4, SNRK, DYRK3, RNF32, JUNB, KCNIP3, CCDC68,CSN3, TRAIP, which are available at the DNASU Plasmid Repository at theBio Design Institute of the Arizona State University, Tempe, Ariz. Inaddition, we also developed a classifier to differentiate patients withBLBC from healthy controls with 33% sensitivity at 98% specificity(Table3).

We designated samples as positive if they exceeded antigen-specificcutoffs for at least 2 of the 13 antigens. Antigen-specific cutoffs wereset to achieve 98% classifier specificity by adjusting the specificityat the antigen level to 98.7%.

A point of novelty is the identification of the 28 antigens that arepotential biomarkers for early detection of basal like breast cancer(Table 1, 2). Many of these 28 antigens have not been previouslyassociated with basal like breast cancer. In addition, we also developeda classifier to differentiate patients with BLBC from healthy controlswith 33% sensitivity at 98% specificity (Table3).

TABLE 1 Training and Validation Statistics for Potential basal-likebreast cancer Biomarkers Training (Cohort1&2: basal, Validation(Cohort3: basal, n = 95; healthy, n = 95) n = 50; healthy, n = 50)Antigen sensitivity specificity cutoffs sensitivity specificity CTAG1B0.213 0.979 1.606 0.200 1.000 CTAG2 0.191 0.979 1.149 0.180 0.960 TRIM210.158 0.979 1.208 0.140 0.860 RNF216 0.110 0.978 1.369 0.043 0.956 MN10.105 0.979 1.311 0.060 0.920 PIP4K2C 0.105 0.979 1.200 0.020 1.000 TP530.084 0.979 3.171 0.200 1.000 ZBTB16 0.084 0.979 1.393 0.040 0.980 TRAIP0.074 0.979 2.682 0.040 0.980 DOK2 0.074 0.979 1.164 0.060 1.000 CSN30.063 0.979 1.955 0.060 0.980 PPHLN1 0.063 0.979 3.394 0.080 1.000TAS2R8 0.063 0.979 1.064 0.080 0.940 SSMEM1 0.063 0.979 1.562 0.0600.960 DYRK3 0.063 0.979 1.462 0.040 0.940 KRT8 0.053 0.979 1.645 0.0600.960 LMO4 0.053 0.979 1.199 0.020 0.980 WBP2NL 0.053 0.979 1.991 0.0600.980 JUNB 0.042 0.979 1.165 0.020 0.960 TSGA13 0.042 0.979 1.313 0.0200.980 PVRL4 0.042 0.979 0.899 0.020 0.920 CCDC68 0.042 0.979 2.438 0.0000.940 BCL2 0.042 0.979 1.160 0.000 1.000 SNRK 0.032 0.979 4.127 0.0200.960 PSRC1 0.032 0.979 1.372 0.120 0.960 KCNIP3 0.032 0.979 0.973 0.0000.960 POU4F1 0.032 0.979 0.992 0.080 0.940 RNF32 0.021 0.979 1.445 0.0400.980

TABLE 2 Performance of 28 antigens in all subtypes of breast cancer.Sensitivity Her2- Antigen basal-like luminal A luminal B enrichedSpecificity CTAG1B 0.208 0.033 0.045 0.056 0.979 CTAG2 0.188 0.000 0.0000.000 0.979 TP53 0.124 0.033 0.000 0.056 0.979 RNF216 0.088 0.133 0.0950.000 0.978 PPHLN1 0.083 0.100 0.182 0.000 0.979 PIP4K2C 0.076 0.1000.091 0.111 0.979 ZBTB16 0.069 0.000 0.000 0.000 0.979 TAS2R8 0.0690.000 0.000 0.056 0.979 WBP2NL 0.069 0.100 0.091 0.000 0.979 DOK2 0.0690.133 0.091 0.056 0.979 PSRC1 0.063 0.033 0.045 0.056 0.979 MN1 0.0620.100 0.000 0.056 0.979 TRAIP 0.062 0.067 0.045 0.000 0.979 CSN3 0.0620.100 0.182 0.000 0.979 TRIM21 0.055 0.033 0.000 0.056 0.979 POU4F10.048 0.033 0.000 0.222 0.979 SSMEM1 0.048 0.033 0.136 0.000 0.979 LMO40.041 0.033 0.000 0.056 0.979 BCL2 0.041 0.033 0.045 0.000 0.979 KRT80.034 0.033 0.000 0.056 0.979 TSGA13 0.034 0.000 0.000 0.056 0.979 PVRL40.034 0.000 0.000 0.000 0.979 SNRK 0.028 0.000 0.045 0.000 0.979 DYRK30.028 0.033 0.045 0.000 0.979 RNF32 0.028 0.033 0.000 0.056 0.979 JUNB0.021 0.000 0.000 0.000 0.979 KCNIP3 0.014 0.033 0.000 0.000 0.979CCDC68 0.007 0.000 0.000 0.000 0.979

TABLE 3 Cutoffs for 13-AAb classifier Antigen Cutoff CTAG1B 1.606 CTAG21.176 TP53 3.171 RNF216 1.459 PPHLN1 3.448 PIP4K2C 1.201 ZBTB16 1.925TAS2R8 1.178 WBP2NL 2.120 DOK2 1.164 PSRC1 1.461 MN1 1.687 TRIM21 5.509

An example of how a patient sample would be handled to detect anddiagnose basal-like breast cancer using one or more of the discoveredbiomarkers in a kit with a suitable detection agent is as follows.

A patient's plasma sample is obtained, and then subsequently tested forautoantibody responses against the proposed protein panels. Briefly,protein targets are produced either freshly in situ or purified ahead oftime, and immobilized on solid surface. A plasma sample is thenincubated with these protein targets. Labeled secondary antibody thatcan recognize human immunoglobulins are used for the signal read out.Accordingly, data such as that shown in FIG. 1 can be obtained and usedto detect and/or diagnose basal-like breast cancer.

Various changes in the details and components that have been describedmay be made by those skilled in the art within the principles and scopeof the invention herein described in the specification and defined inthe appended claims. Therefore, while the present invention has beenshown and described herein in what is believed to be the most practicaland preferred embodiments, it is recognized that departures can be madetherefrom within the scope of the invention, which is not to be limitedto the details disclosed herein but is to be accorded the full scope ofthe claims so as to embrace any and all equivalent processes andproducts.

What is claimed is:
 1. A method for detection of basal-like breastcancer in a patient sample harboring one or more antigens, comprising:detecting a tumor antigen biomarker in said patient sample, wherein thebiomarker is selected from the antigens: P53 (TP53), NY-ESO-1 (CTAG1B),NY-ESO-2 (CTAG2), RNF216, PPHLN1, PIP4K2C, ZBTB16, TAS2R8, WBP2NL, DOK2,PSRC1, MN1, TRIM21, POU4F1, SSMEM1, LMO4, BCL2, KRT8, TSGA13, PVRL4,SNRK, DYRK3, RNF32, JUNB, KCNIP3, CCDC68, CSN3, TRAIP.
 2. The method ofclaim 1, wherein said detecting comprises utilizing an enzyme-linkedimmunosorbant assay (ELISA).
 3. The method of claim 1, furthercomprising detecting at least two of said tumor antigen biomarkers andcomparing a level of detection to predetermined sensitivity andspecificity threshold to determine if basal-like breast cancer isindicated.
 4. A basal-like breast cancer diagnostic test kit, comprisingan antibody against a tumor antigen biomarker selected from the groupconsisting of: P53 (TP53), NY-ESO-1 (CTAG1B), NY-ESO-2 (CTAG2), RNF216,PPHLN1, PIP4K2C, ZBTB16, TAS2R8, WBP2NL, DOK2, PSRC1, MN1, TRIM21,POU4F1, SSMEM1, LMO4, BCL2, KRT8, TSGA13, PVRL4, SNRK, DYRK3, RNF32,JUNB, KCNIP3, CCDC68, CSN3, TRAIP, and a suitable detection agent. 5.The kit of claim 4, wherein an antibody against each of said tumorantigen biomarkers is present in said kit.
 6. A method for diagnosingbasal-like breast cancer from a patient sample harboring one or moreantigens, comprising: a) detecting a tumor antigen biomarker in saidpatient sample, wherein the biomarker is selected from the antigens: P53(TP53), NY-ESO-1 (CTAG1B), NY-ESO-2 (CTAG2), RNF216, PPHLN1, PIP4K2C,ZBTB16, TAS2R8, WBP2NL, DOK2, PSRC1, MN1, TRIM21, POU4F1, SSMEM1, LMO4,BCL2, KRT8, TSGA13, PVRL4, SNRK, DYRK3, RNF32, JUNB, KCNIP3, CCDC68,CSN3, TRAIP; and b) comparing a detection result of step a) with acontrol.